Application of benzylselenenitrile in preparation of medicine for preventing and / or treating pancreatic cancer

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of benzyl selenenitrile in preparation of a medicine for preventing and / or treating pancreatic cancer. The benzylselenenitrile can effectively inhibit in-vivo growth of pancreatic cancer by improving immune cell infiltration in a pancreatic cancer tumor microenvironment and a killing function of anti-tumor cells such as CD8 + T cells, so that the effect of preventing and / or treating the pancreatic cancer is achieved. Experimental results show that the structures of the liver, spleen, kidney and lung of the mouse subjected to BSC intraperitoneal injection treatment have no significant difference from those of a control mouse, and the proliferation capacity of bone marrow cells also has no significant difference, so that the BSC does not generate visceral organ and bone marrow toxicity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of benzene selenocyanide in the preparation of drugs for the prevention and / or treatment of pancreatic cancer. Background Technology

[0002] Pancreatic cancer is a highly lethal malignant tumor. According to global cancer statistics, the incidence of pancreatic cancer is increasing year by year, and it is currently the seventh leading cause of cancer-related death worldwide. It is projected that by 2040, pancreatic cancer will surpass colorectal cancer, becoming the second leading cause of cancer-related death after lung cancer. Furthermore, except for a few cases caused by germline mutations or known risk factors (such as mucinous cystic lesions and chronic pancreatitis), most patients have insidious early symptoms and lack clear risk factors, making early detection extremely difficult. At the same time, due to the lack of effective early biomarkers, screening and diagnosis in the early stages of the disease are very difficult in clinical practice. Therefore, pancreatic cancer is usually discovered only at an advanced stage, at which point most patients often have metastasized to other organs such as the liver. Currently, the five-year survival rate for pancreatic cancer is less than 10%, the lowest among all cancers. Treatment methods for pancreatic cancer mainly include surgical resection, radiotherapy, chemotherapy, and targeted therapy, among which surgical resection is currently the most effective treatment. However, only about 20% of patients meet the criteria for resection in the early stages, and the tumor recurrence rate is as high as 62% within 12 months after resection, with a 5-year survival rate of less than 30%. For patients with advanced pancreatic cancer who are not candidates for surgery, common treatments include combination chemotherapy with drugs such as paclitaxel and gemcitabine, but the efficacy is limited and accompanied by significant side effects, including organ and bone marrow toxicity.

[0003] Immune cells are involved in maintaining systemic homeostasis and regulating disease progression. However, pancreatic cancer, with its dense microenvironment formed by high-density connective tissue and extracellular matrix, hinders immune cell infiltration. Furthermore, the tumor microenvironment contains numerous factors that suppress immune cells. Avoiding significant toxic side effects on other organs poses a major challenge to targeted therapy for pancreatic cancer. Therefore, there is an urgent need to provide a safer drug for treating pancreatic cancer. Summary of the Invention

[0004] The purpose of this invention is to provide the use of benzene selenocyanide in the preparation of drugs for the prevention and / or treatment of pancreatic cancer; benzene selenocyanide has the effect of preventing and / or treating pancreatic cancer, and does not produce organ and bone marrow toxicity when used in vivo.

[0005] This invention provides the use of benzene selenonitrile in the preparation of drugs for the prevention and / or treatment of pancreatic cancer.

[0006] As a preferred embodiment, the drug comprises: benzene selenocyanide and pharmaceutically acceptable excipients.

[0007] As a preferred embodiment, the effective concentration of benzene selenonitrile in the drug is 5-10 mg / kg mouse based on mouse body weight.

[0008] As a preferred embodiment, the dosage form of the drug includes at least one of the following: injection, powder, granule, premix, and oral liquid.

[0009] As a preferred embodiment, the injection is administered via intraperitoneal injection.

[0010] As a preferred embodiment, the excipients include: DMSO solution and / or PBS buffer.

[0011] As a preferred embodiment, the efficacy of the drug includes at least one of the following: (1) inhibiting tumor growth; (2) promoting immune cell infiltration; (3) enhancing immune response; and (4) having no toxic side effects on organs and bone marrow.

[0012] As a preferred embodiment, the immune cells include at least one of the following: CD45 + Immune cells, macrophages, and T cells.

[0013] As a preferred embodiment, the enhanced immune effect includes at least one of the following: (1) increasing CD4 + The proportion of cells, (2) increases CD8 + (3) Promote the expression of T cell effector factors.

[0014] As a preferred embodiment, the organ includes at least one of the following: liver, spleen, lung, and kidney.

[0015] Beneficial effects: This invention provides the application of benzyl selenocyanate in the preparation of drugs for the prevention and / or treatment of pancreatic cancer; the benzyl selenocyanate (hereinafter referred to as BSC) enhances the infiltration of immune cells in the pancreatic cancer tumor microenvironment and CD8+. + The killing function of anti-tumor cells such as T cells can effectively inhibit the growth of pancreatic cancer in vivo, thereby achieving the effect of preventing and / or treating pancreatic cancer. Furthermore, experimental results show that the structures of the liver, spleen, kidneys, and lungs of mice treated with intraperitoneal injection of BSC were not significantly different from those of control mice, indicating that BSC does not produce organ toxicity. Attached Figure Description

[0016] Figure 1 The experimental flowchart shows the effect of BSC on tumor growth in mice with pancreatic cancer. Figure 2 The graph shows the results of BSC on changes in tumor volume in mice with pancreatic cancer; Figure 3 This is a photograph showing the effect of BSC on tumor volume in mice with pancreatic cancer. Figure 4 The graph shows the effect of BSC on the survival rate of mice with pancreatic cancer. Figure 5 This is a graph showing the effect of BSC on immune cells within pancreatic cancer tumors. Figure 6 The figure shows the effects of BSC administration on abdominal organs. Detailed Implementation

[0017] This invention provides the use of benzene selenonitrile in the preparation of drugs for the prevention and / or treatment of pancreatic cancer.

[0018] The benzene selenocyanide of this invention has the effect of preventing and / or treating pancreatic cancer, and does not produce organ and bone marrow toxicity when used in vivo. As a preferred embodiment, the benzene selenocyanide is a white solid powder; the structural formula of the benzene selenocyanide is as shown in Formula I below: Formula I.

[0019] In a preferred embodiment, the drug comprises benzene selenocyanide and pharmaceutically acceptable excipients. The effective concentration of benzene selenocyanide in the drug of the present invention, based on mouse body weight, can be any value within the range of 5-10 mg / kg mice, for example, 5, 7.5, or 10 mg / kg mice. In a preferred embodiment, the dosage form comprises at least one of the following: injection, powder, granule, premix, and oral liquid. In a preferred embodiment, the injection is administered via intraperitoneal injection. In a preferred embodiment, the excipients comprise DMSO (dimethyl sulfoxide) solution and / or PBS buffer.

[0020] In one embodiment, the efficacy of the drug includes at least one of the following: (1) Inhibits tumor growth; (2) Promotes the infiltration of immune cells; the immune cells include at least one of the following: CD45 + Immune cells, macrophages, and T cells; (3) Enhanced immune response; the enhanced immune response includes at least one of the following: increasing CD4 + Cell ratio; increase CD8 + The proportion of T cells; promoting the expression of T cell effector factors; (4) It has no toxic side effects on organs and bone marrow; the organs include at least one of the following: liver, spleen, lung and kidney.

[0021] The selenocyanide described in this invention enhances the infiltration of immune cells within the pancreatic cancer tumor microenvironment and CD8+. +The killing function of anti-tumor cells such as T cells can effectively inhibit the growth of pancreatic cancer in vivo, thereby achieving effective prevention and / or treatment of pancreatic cancer. Furthermore, experimental results show that the structure of the liver, spleen, kidneys, and lungs of mice treated with intraperitoneal injection of BSC was not significantly different from that of control mice, and the proliferative capacity of bone marrow cells was also not significantly different, indicating that BSC does not produce organ and bone marrow toxicity.

[0022] To further illustrate the present invention, the application of the selenocyanide provided by the present invention in the preparation of drugs for the prevention and / or treatment of pancreatic cancer is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] Experimental materials Benzyl selenocyanate (BSC), purchased from MCE (product number: HY-131991), with a purity of 99.84%, is a white solid powder. Before use, it was dissolved in DMSO (dimethyl sulfoxide) to a stock solution of 25 mg / mL, and then diluted with PBS buffer to 2.5 mg / mL to obtain the BSC solution, which was used for subsequent intraperitoneal injection in mice.

[0024] The chemical structural formula is: .

[0025] The mouse-derived pancreatic cancer cells PANC02 (hereinafter referred to as PANC02 cells) were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0026] The flow cytometry antibodies included: Alexa Fluor® 700 anti-mouse CD45 antibody, PE-CY7 anti-mouse CD11b antibody, PE anti-mouse F4 / 80 antibody, BV650 anti-mouse CD86 antibody, BV605 anti-mouse CD4 antibody, APC-CY7 anti-mouse CD8 antibody, PE anti-mouse IFNg antibody, and APC anti-mouse TNFa antibody; all purchased from Biolegend.

[0027] The wild-type mouse strain was C57BL / 6J, housed in an SPF-grade animal facility at an ambient temperature of 25±1℃ and a relative humidity of 50%~60%, with a 12h / 12h diurnal cycle. The mice had free access to water and food, with sterilized tap water as drinking water, cobalt-60 irradiated sterilized feed, and sterilized corn cob bedding.

[0028] The flow cytometer used was a Beckman Cytoflex flow cytometer.

[0029] All other reagents or instruments used without a specified manufacturer are commercially available products.

[0030] Example 1 The effect of BSC on tumor growth in mice with pancreatic cancer, experimental procedure as follows: Figure 1 As shown.

[0031] 1) PANC02 cells were cultured in DMEM complete medium. After the cells were in good condition and in the logarithmic growth phase, they were digested with 0.25% trypsin (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C0201). After centrifugation, the cell concentration was adjusted to 3×10⁻⁶ cells in pre-cooled DMEM complete medium at 4°C. 7 The concentration of cells / mL was increased, and then an equal volume of matrix gel was added and mixed thoroughly to obtain pancreatic cancer cells to be inoculated. Each mouse was inoculated with 100 μL of pancreatic cancer cells (50 μL of pancreatic cancer cell fluid and 50 μL of matrix gel). Before inoculation, the hair on the side of the mouse to be inoculated was shaved, and the area was disinfected with 70% ethanol. Then, pancreatic cancer cells were inoculated using a 1 mL sterile syringe.

[0032] 2) Mice inoculated with pancreatic cancer cells were divided into two groups: the PBS group (control group) and the BSC group (experimental group). Eight mice were set in each group for survival statistics, and five mice were set in each group for flow cytometry analysis.

[0033] 3) BSC group: On days 4, 8, 12 and 16 after inoculation with pancreatic cancer cells, mice were intraperitoneally injected with 100 μL of BSC solution with a concentration of 2.5 mg / mL. The BSC concentration was 5 mg, and the weight of each mouse was about 25 mg. Therefore, the dosage of BSC was 10 mg / kg of mice based on the weight of the mice.

[0034] PBS group: Mice were intraperitoneally injected with 200 μL of PBS buffer on days 4, 8, 12 and 16 after inoculation with pancreatic cancer cells.

[0035] 4) Starting from day 5 after inoculation with pancreatic cancer cells, the length and width of the tumor were measured every 5 days. The tumor volume was calculated using the formula V = length × width × width / 2. A total of 8 mice were treated in each group. Each column shows the tumor volume of a single mouse measured on the corresponding modeling day. The results are shown in […]. Figure 2 See Table 1. Mice were sacrificed 18 days after inoculation with pancreatic cancer cells, and the tumors were removed using forceps. The results are shown in Table 1. Figure 3 For volumes V > 1500 mm² 3 The mice were euthanized. The number of surviving mice was measured every 5 days, and the survival rate was calculated using the formula: Survival rate = (Number of surviving mice / Total number of mice) × 100%. The results are shown below. Figure 4 And Table 2.

[0036] Table 1. Effects of BSC on pancreatic cancer tumor volume in mice (mm) 3 )change

[0037] Table 2. Effect of BSC on survival rate in mice with pancreatic cancer.

[0038] from Figures 2-4 As shown in Tables 1 and 2, intraperitoneal injection of BSC solution can significantly inhibit tumor growth in mice and also improve the survival time of mice.

[0039] Example 2 Effects of BSC on immune cells within pancreatic cancer tumors Steps 1) to 3) are performed according to Example 1.

[0040] 4) Based on the volume of digestion fluid, the mixed digestion solution consisted of 1 mg / mL collagenase II, 1 mg / mL collagenase IV, 20 μg / mL DNase I, and 2% (v / v) fetal bovine serum added to the digestion fluid. Collagenase II and collagenase IV were purchased from Thermo Fisher Scientific as powder and were prepared as a 50 mg / kg stock solution with PBS before use. DNase I was purchased from Roche and was also prepared as a 5 mg / mL stock solution with PBS before use, and then added to the digestion fluid according to the working concentration.

[0041] Pancreatic cancer tissue was harvested on day 18 after cell inoculation with pancreatic cancer cells. The tissue was washed with pre-cooled PBS buffer, minced, and mixed with digestion solution. The mixture was incubated in a shaker at 37°C for 1 hour. After filtration through a cell sieve, the filtrate was collected to obtain a cell suspension, which was then centrifuged. The supernatant was collected to obtain the cell solution. Mononuclear cells were obtained by separating the cell solution using Percoll lymphocyte separation medium and centrifuged at 500 g / min for 10 minutes. During this period, staining solutions were prepared. PBS was added to 1.5 mL EP tubes containing 50 μL of chromosomes per mononuclear cell. The corresponding flow cytometry antibodies were added to the PBS: Alexa Fluor® 700 anti-mouse CD45 antibody, PE-CY7 anti-mouse CD11b antibody, PE anti-mouse F4 / 80 antibody, BV650 anti-mouse CD86 antibody, BV605 anti-mouse CD4 antibody, APC-CY7 anti-mouse CD8 antibody, PE anti-mouse IFNγ antibody, and APC anti-mouse TNFα antibody. The added volume of the 0.2 mg / mL flow cytometry antibody was 1 / 400 (v / v) of the mononuclear cell volume, resulting in a working staining solution with a concentration of 0.5 μg / mL. The mononuclear cell pellet obtained after centrifugation was then resuspended in the working staining solution and stained at 4°C in the dark for 30 minutes. After 30 minutes, the mononuclear cells were centrifuged again, resuspended in 200 μL PBS, and analyzed by flow cytometry. The results are shown below. Figure 5 And Table 3.

[0042] Table 3 Flow cytometry analysis results

[0043] from Figure 5 As shown in Table 3, BSC significantly promoted CD45. + Immune cells infiltrate within the tumor and increase pro-inflammatory CD86. + Macrophages, IFNγ + and TNFα + CD4 + and CD8 + The proportion of T cells. CD45 + The content of immune cells represents the proportion of all immune cells in tumor tissue, indicating that BSC drug treatment can promote the infiltration of immune cells into the tumor. Further analysis of pro-inflammatory immune cells within the tumor, namely CD86... +Macrophages and IFNγ / TNFα-producing T cells showed a significant increase in pro-inflammatory immune cells, indicating that BSCs promoted immune effector function. Therefore, these results suggest that in vivo administration of BSCs inhibited tumor growth in mice and promoted immune cell infiltration and immune effector function.

[0044] Example 3 Side effects of BSC administration on abdominal organs Steps 1) to 3) are performed according to Example 1.

[0045] 4) On day 18 after inoculation of pancreatic cancer cells, liver, spleen, lung, and kidney tissues were collected and placed in 4% paraformaldehyde. After dehydration, paraffin embedding, sectioning, and dewaxing, the tissues were stained with hematoxylin-eosin (HE) and analyzed under a light microscope. The results are shown in [Figure number missing]. Figure 6 .

[0046] from Figure 6 It can be seen that intraperitoneal injection of BSC has no obvious toxic side effects on the liver, spleen, lungs, and kidneys. Specifically, the tissue structure and immune infiltration of the organs are similar to those of the PBS control group.

[0047] Therefore, benzebinol (BSC) has the effect of preventing and / or treating pancreatic cancer, and does not produce organ and bone marrow toxicity when used in vivo.

[0048] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of benzene selenonitrile in the preparation of drugs for the prevention and / or treatment of pancreatic cancer.

2. The application according to claim 1, characterized in that, The drug comprises: selenocyanide and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, Based on mouse body weight, the effective concentration of benzene selenonitrile in the drug is 5-10 mg / kg mouse.

4. The application according to claim 3, characterized in that, The dosage form of the drug includes at least one of the following: injection, powder, granule, premix, and oral liquid.

5. The application according to claim 3, characterized in that, The injectable includes: intraperitoneal injection.

6. The application according to claim 2, characterized in that, The excipients include: DMSO solution and / or PBS buffer.

7. The application according to any one of claims 1 to 6, characterized in that, The efficacy of the drug includes at least one of the following: (1) inhibiting tumor growth; (2) promoting immune cell infiltration; (3) enhancing immune response; and (4) having no toxic side effects on organs and bone marrow.

8. The application according to claim 7, characterized in that, The immune cells include at least one of the following: CD45 + Immune cells, macrophages, and T cells.

9. The application according to claim 7, characterized in that, The enhanced immune effect includes at least one of the following: (1) increasing CD4 + (2) Increase the proportion of T cells; + (3) Promote the expression of T cell effector factors.

10. The application according to claim 7, characterized in that, The organs include at least one of the following: liver, spleen, lungs, and kidneys.

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